摘要
目的 探讨电穿孔技术介导的重组质粒体内转染兔下颌骨牵引成骨的可行性。方法以新西兰大白兔为实验动物模型，于术后3d开始下颌骨牵引，每天0．8mm，连续牵引7d后，将实验动物分为3组：质粒＋电穿孔组（A组），质粒组（B组），生理盐水组＋电穿孔组（C组）。各组动物分别于注射后3h及1、3、7、14d处死，切取牵引区组织0．4cm×0．4cm行冰冻切片检查，采用荧光显微镜观察绿色荧光蛋白（GFP）表达以检测外源基因的表达。检测兔血清肝功能指标丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）和。肾功能指标尿素氮（BUN）、肌酐（Scr）和心、肝、肾组织学检查。结果A组转染新西兰大白兔，3h可观察到GFP的表达，1d时GFP的表达增强，3d时GFP的表达最强，其后开始逐渐下降，7d后GFP的表达减少，14d仍可观察到微弱GFP的表达。B组的GFP的表达时限与A组相同，但各时相点的GFP的表达强度明显弱于A组，C组在各时间段均未观察到GFP的表达。3组肝、肾功能指标两两比较差异无统计学意义（P〉0．05）。结论电穿孔技术介导的带有荧光标记的重组质粒体内转染，可在兔下颌骨牵引区组织内表达，电穿孔能明显提高重组质粒的体内转染效率，提示电穿孔技术介导的重组质粒体内转染兔下颌骨牵引成骨的动物模型是可行的，用于体内试验是安全的。
Objective To evaluate the feasibility of electroporation-mediated transfection of recombinant plasmid to mandibular distraction area of rabbit in vivo. Methods New-Zeland rabbit were employed. The mandible was distracted 3 days after operation at a rate of 0.8 mm per day for 7 days. The rabbits were randomly divided into 3 groups as group A （recombinant plasmid plRES-VEGF165-EGFP）, group B（recombinant plasmid plRES-VEGF165-EGFP） and group C（normal saline）. The rabbits were sacrified at 3 hours, 1, 3, 7 and 14 d after injection respectively. The tissue at the distraction area was taken out for frozen section. The gene expression was assessed by the detection of expression of green fluorescence protein （GFP） using fluorescence microscope. The liver and kidney function test（ALT, AST, BUN, Scr） and the histological examination of heart, liver and kidney were also performed. Results GFP was seen in the distraction area in group A and group B 3 hours after injection, which increased at the 1st day, reached peak value at the 3rd day, decreased at the 7th day and was very lower at 14th day. The GFP expression was much stronger in group A than in group B. GFP was not expressed in group C. There was no statistical difference in the concentration of ALT, AST, BUN and Scr in serum of rabbits among the three groups. Conclusions Eleetroporation-mediated transfection of recombinant plasmid can be expressed in the distraction area of rabbits, and there was no toxity to the liver and kidney of rabbits. Eleetroporation could obviously improve transfection efficiency in vivo. It indicates that electroporation-mediated transfection of recombinant plasmid to distraction area tissue of rabbits is feasible.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2009年第4期280-283,共4页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金资助项目（30600653）,四川省应用基础研究计划项目（2006J13-128）,四川省卫生厅科研课题（060044）
关键词
电穿孔
基因疗法
骨生成
牵张
Eleetroporation
Gene therapy
Osteogenesis, distraction
